The objective of this proposal is to understand the dynamic and structural properties of chromosomes and the spindle during mitosis. Kinetochores are dynamic protein-DNA assemblies situated along eukaryotic chromosomes that are essential for linking chromosomal DNA to microtubules of the mitotic spindle. A breakdown in the fidelity of chromosome segregation has been documented in many types of tumor cells and is likely to be an initiating event in tumor progression. Chromosomal aneuploidy will accelerate tumor development by increasing the rate at which recessive mutations in tumor suppressor genes are uncovered. The identification of kinetochore proteins is one of many critical steps toward understanding mechanisms of chromosome segregation. The key to understanding kinetochore function and chromosome segregation will be the elucidation of mechanisms that link kinetochores to dynamic microtubules. With the advent of live cell fluorescence imaging techniques in budding yeast, the completion of the yeast genome project, and efforts to describe the network of protein-protein interactions, we are poised to address mechanistic processes by which kinetochores capture microtubules, attain bipolar orientation, and promote chromosome dynamics and segregation. The specific objectives are to determine the role of kinetochore components in governing kinetochore microtubule dynamics and kinetochore attachment/detachment, quantitative the dynamic properties of kinetochore proteins, and determine how these dynamic assemblies interact with microtubules to generate a mechanically sound linkage. We will determine the structural requirements that allow centromere flanking DNA to become highly extended upon microtubule attachment, and whether chromosome stretching contributes to the spindle checkpoint. Our approach entails chromosome engineering, analysis of chromosome and protein dynamics by quantitative fluorescence microscopy and genetic manipulation of kinetochore components.